Moxonidine modulates cytokine signalling and effects on cardiac cell viability.

Regression of left ventricular hypertrophy and improved cardiac function in SHR by the centrally acting imidazoline I1-receptor agonist, moxonidine, are associated with differential actions on circulating and cardiac cytokines. Herein, we investigated cell-type specific I1-receptor (also known as nischarin) signalling and the mechanisms through which moxonidine may interfere with cytokines to affect cardiac cell viability. Studies were performed on neonatal rat cardiomyocytes and fibroblasts incubated with interleukin (IL)-1ß (5 ng/ml), tumor necrosis factor (TNF)-α (10 ng/ml), and moxonidine (10(-7) and 10(-5) M), separately and in combination, for 15 min, and 24 and 48 h for the measurement of MAPKs (ERK1/2, JNK, and p38) and Akt activation and inducible NOS (iNOS) expression, by Western blotting, and cardiac cell viability/proliferation and apoptosis by flow cytometry, MTT assay, and Live/Dead assay. Participation of imidazoline I1-receptors and the signalling proteins in the detected effects was identified using imidazoline I1-receptor antagonist and signalling protein inhibitors. The results show that IL-1ß, and to a lower extent, TNF-α, causes cell death and that moxonidine protects against starvation- as well as IL-1ß -induced mortality, mainly by maintaining membrane integrity, and in part, by improving mitochondrial activity. The protection involves activation of Akt, ERK1/2, p38, JNK, and iNOS. In contrast, moxonidine stimulates basal and IL-1ß-induced fibroblast mortality by mechanisms that include inhibition of JNK and iNOS. Thus, apart from their actions on the central nervous system, imidazoline I1-receptors are directly involved in cardiac cell growth and death, and may play an important role in cardiovascular diseases associated with inflammation.

An "I" on cardiac hypertrophic remodelling: imidazoline receptors and heart disease.

The centrally-acting sympatholytic imidazoline compound, moxonidine, prevents the development of left ventricular hypertrophy and attenuates maladaptive proliferative signalling as well as downstream apoptotic pathways in spontaneously hypertensive rat and cardiomyopathic hamster hearts. The actions are selectively mediated by imidazoline type-1 receptor (I(1)-receptor, also named nischarin), nonadrenergic neurotransmitter receptors mainly found in the brainstem medulla. We identified cardiac I(1)-receptors/nischarin and showed that they are upregulated in cardiovascular disorders, and are functional without the central nervous system's contribution. Molecular characterization revealed that I(1)-receptor/nischarin has a unique structure with multifunctional domains allowing it to perform a number of cell signalling roles as a scaffolding protein. Nischarin has been associated with integrin α5 and inhibition of Rac1 and was shown to interact with insulin receptor substrates. However, very little is known about cardiac I(1)-receptor/nischarin and its role(s) in normal physiology and pathophysiology, specifically in cardiac remodelling. Our studies have shown that I(1)-receptor is expressed in cardiac fibroblasts and myocytes and that in vitro I(1)-receptor activation inhibits norepinephrine-induced cardiomyocyte apoptosis and fibroblast proliferation, through differential effects on mitogen-activated protein kinases and Akt. Accordingly, apart from centrally-mediated sympatholytic function, I(1)-receptor in the heart may control cell growth and death. I(1)-receptor may be implicated in cardiac remodelling and dysfunction, through the inhibition of apoptotic pathways and/or activation of survival pathways, in a cell-specific manner. Identification of the cardioprotective mechanisms of cardiac I(1)-receptor could result in specifically-tailored cell/gene-driven I(1)-receptor treatments, and/or treatments that target cardiac I(1)-receptor, which could eventually be important for patients with hypertrophic heart disease.

Functional and molecular effects of imidazoline receptor activation in heart failure.

AIMS: Heart failure is a progressive deterioration in heart function associated with overactivity of the sympathetic nervous system. The benefit of inhibition of sympathetic activity by moxonidine, a centrally acting imidazoline receptor agonist, was questioned based on the outcome of a failing clinical trial. The following studies measured cardiac structure and hemodynamics and mechanisms underlying moxonidine-induced changes, in cardiomyopathic hamsters, where the stage of the disease, dose, and compliance were controlled. MAIN METHODS: Male BIO 14.6 hamsters (6 and 10 months old, with moderate and advanced heart failure, respectively) received moxonidine at 2 concentrations: low (2.4 mg/kg/day) and high (9.6 mg/kg/day), or vehicle, subcutaneously, for 1month. Cardiac function was measured by echocardiography, plasma and hearts were collected for histological determination of fibrosis and apoptosis, as well as for measurement cytokines by Elisa and cardiac proteins by Western blotting. KEY FINDINGS: Compared to age-matched vehicle-treated BIO 14.6, moxonidine did not reduce blood pressure but significantly reduced heart rate and improved cardiac performance. Moxonidine exerted anti-apoptotic effect with differential inflammatory/anti-inflammatory responses that culminate in attenuated cardiac apoptosis and fibrosis and altered protein expression of collagen types. Some effects were observed regardless of treatment onset, although the changes were more significant in the younger group. Interestingly, moxonidine resulted in upregulation of cardiac imidazoline receptors. SIGNIFICANCE: These studies imply that in addition to centrally mediated sympathetic inhibition, the effects of moxonidine may, at least in part, be mediated by direct actions on the heart. Further investigation of imidazolines/imidazoline receptors in cardiovascular diseases is warranted.

Hemodynamic and cardiac effects of chronic eprosartan and moxonidine therapy in stroke-prone spontaneously hypertensive rats.

The renin-angiotensin and sympathetic nervous systems play critical interlinked roles in the development of left ventricular hypertrophy, fibrosis, and dysfunction. These studies investigated the hemodynamic and cardiac effects of monoblockade and coblockade of renin-angiotensin and sympathetic nervous systems. Stroke-prone spontaneously hypertensive rats (16 weeks old; male; n=12 per group) received the sympatholytic imidazoline compound, moxonidine (2.4 mg/kg per day); the angiotensin-receptor blocker eprosartan (30 mg/kg per day), separately or in combination; or saline vehicle for 8 weeks, SC, via osmotic minipumps. Blood pressure and heart rate were continuously measured by radiotelemetry. After 8 weeks, in vivo cardiac function and structure were measured by transthoracic echocardiography and a Millar conductance catheter, and the rats were then euthanized and blood and heart ventricles collected for various determinations. Compared with vehicle, the subhypotensive dose of moxonidine resulted in lower (P<0.01) heart rate, left ventricular hypertrophy, cardiomyocyte cross-sectional area, interleukin 1 beta, tumor necrosis factor-alpha, and mRNA for natriuretic peptides. Eprosartan reduced pressure (P<0.01), as well as extracellular signal-regulated kinase (ERK) 44 phosphorylation, Bax/Bcl-2, and collagen I/III, and improved left ventricular diastolic function (P<0.03). Combined treatment resulted in greater reductions in blood pressure, heart rate, left ventricular hypertrophy, collagen I/III, and inhibited inducible NO synthase and increased endothelial NO synthase phosphorylation, as well as reduced left ventricular anterior wall thickness, without altering the other parameters. Thus, in advanced hypertension complicated with cardiac fibrosis, sympathetic inhibition and angiotensin II blockade resulted in greater reduction in blood pressure and heart rate, inhibition of inflammation, and improved left ventricular pathology but did not add to the benefits of angiotensin II blockade on cardiac function.

The role of natriuretic peptide receptor-A signaling in unilateral lung ischemia-reperfusion injury in the intact mouse.

Ischemia-reperfusion (IR) causes human lung injury in association with the release of atrial and brain natriuretic peptides (ANP and BNP), but the role of ANP/BNP in IR lung injury is unknown. ANP and BNP bind to natriuretic peptide receptor-A (NPR-A) generating cGMP and to NPR-C, a clearance receptor that can decrease intracellular cAMP. To determine the role of NPR-A signaling in IR lung injury, we administered the NPR-A blocker anantin in an in vivo SWR mouse preparation of unilateral lung IR. With uninterrupted ventilation, the left pulmonary artery was occluded for 30 min and then reperfused for 60 or 150 min. Anantin administration decreased IR-induced Evans blue dye extravasation and wet weight in the reperfused left lung, suggesting an injurious role for NPR-A signaling in lung IR. In isolated mouse lungs, exogenous ANP (2.5 nM) added to the perfusate significantly increased the filtration coefficient sevenfold only if lungs were subjected to IR. This effect of ANP was also blocked by anantin. Unilateral in vivo IR increased endogenous plasma ANP, lung cGMP concentration, and lung protein kinase G (PKG(I)) activation. Anantin enhanced plasma ANP concentrations and attenuated the increase in cGMP and PKG(I) activation but had no effect on lung cAMP. These data suggest that lung IR triggered ANP release and altered endothelial signaling so that NPR-A activation caused increased pulmonary endothelial permeability.

Receptors involved in moxonidine-stimulated atrial natriuretic peptide release from isolated normotensive rat hearts.

Imidazoline I1-receptors are present in the heart and may be involved in atrial natriuretic peptide (ANP) release. The following studies investigated whether moxonidine (an antihypertensive imidazoline I1-receptor and alpha2-adrenoceptor agonist) acts directly on the heart to stimulate ANP release, and to characterize the receptor type involved in this action. Perfusion of rat (200-225 g) isolated hearts with moxonidine (10(-6) and 10(-5) M), for 30 min, resulted in ANP release (83+/-29 and 277+/-70 ng/30 min, above basal, respectively), significantly (P<0.01) different from perfusion with buffer (-6+/-31 ng/30 min). ANP release stimulated by moxonidine (10(-6) M) was inhibited by co-perfusion with the antagonists, AGN192403 (imidazoline I1-receptor), phenoxybenzamine (alpha2>alpha1-adrenoceptors), and prazosin (alpha1>alpha2-adrenoceptors), but increased by rauwolscine (alpha2-adrenoceptors). Perfusion with 10(-5) M brimonidine (full alpha2-adrenoceptor agonist) inhibited moxonidine-stimulated ANP release. Similarly, moxonidine (10(-6) M) tended to reduce coronary flow, but significantly increased coronary flow in the presence of brimonidine, which was vasoconstrictive when perfused alone. Coronary flow was reduced by 10(-5) M each, brimonidine>clonidine>moxonidine; while similar bradycardia was observed with clonidine and moxonidine, but not with brimonidine. In conclusion, these results argue in favor of moxonidine acting primarily on imidazoline I1-receptors to release ANP, with both alpha2-adrenoceptor and imidazoline I1-receptors exerting inhibitory inter-relation. In contrast, the coronary vasodilatory effect of moxonidine requires full activation of alpha2-adrenoceptor. The sympatholytic and ANP-releasing effects of moxonidine appear to be mediated by cardiac imidazoline receptors that may be differentially localized. Most importantly, moxonidine can stimulate ANP release from the heart without contribution of the central nervous system.

Natriuretic peptides as therapeutic targets.

Natriuretic peptides (atrial natriuretic peptide, brain natriuretic peptide and C-type natriuretic peptide) are cardiac and vascular peptides with vasodilatory, diuretic, natriuretic, anti-inflammatory, antifibrotic and antimitogenic actions. Natriuretic peptides are implicated in normal pressure and volume homeostasis and in the defence against excessive increases in overload-related factors, vasopressive and cardiotoxic factors and their impact on the heart, blood vessels and brain. Genetic manipulation studies confirmed the importance of natriuretic peptides in these functions. Natriuretic peptides are metabolised by NPR-C (clearance receptors) and by enzymatic degradation by neutral endopeptidase. Natriuretic peptide levels (mainly brain natriuretic peptide) correlate with left ventricular hypertrophy and with the severity of heart failure, and are reduced by effective treatment, thus used as diagnostic and prognostic tools. Based on the multiple protective effects of natriuretic peptides, pharmacological therapy has been approved and includes potentiating natriuretic peptide levels by intravenous infusion or by inhibition of endogenous natriuretic peptide degradation. Because each approach has its limitations, the field remains open for improvement.

Urinary responses to acute moxonidine are inhibited by natriuretic peptide receptor antagonist.

We have previously shown that acute intravenous injections of moxonidine and clonidine increase plasma atrial natriuretic peptide (ANP), a vasodilator, diuretic and natriuretic hormone. We hypothesized that moxonidine stimulates the release of ANP, which would act on its renal receptors to cause diuresis and natriuresis, and these effects may be altered in hypertension. Moxonidine (0, 10, 50, 100 or 150 microg in 300 microl saline) and clonidine (0, 1, 5 or 10 microg in 300 microl saline) injected intravenously in conscious normally hydrated normotensive Sprague-Dawley rats (SD, approximately 200 g) and 12-14-week-old Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) dose-dependently stimulated diuresis, natriuresis, kaliuresis and cGMP excretion, with these effects being more pronounced during the first hour post-injection. The actions of 5 microg clonidine and 50 microg moxonidine were inhibited by yohimbine, an alpha2-adrenoceptor antagonist, and efaroxan, an imidazoline I1-receptor antagonist. Moxonidine (100 microg) stimulated (P<0.01) diuresis in SHR (0.21+/-0.04 vs 1.16+/-0.06 ml h(-1) 100 g(-1)), SD (0.42+/-0.06 vs 1.56+/-0.19 ml h(-1) 100 g(-1)) and WKY (0.12+/-0.04 vs 1.44+/-0.21 ml h(-1) 100 g(-1)). Moxonidine-stimulated urine output was lower in SHR than in SD and WKY. Moxonidine-stimulated sodium and potassium excretions were lower in SHR than in SD, but not WKY, demonstrating an influence of strain but not of pressure. Pretreatment with the natriuretic peptide antagonist anantin (5 or 10 microg) resulted in dose-dependent inhibition of moxonidine-stimulated urinary actions. Anantin (10 microg) inhibited (P<0.01) urine output to 0.38+/-0.06, 0.12+/-0.01, and 0.16+/-0.04 ml h(-1) 100 g(-1) in SD, WKY, and SHR, respectively. Moxonidine increased (P<0.01) plasma ANP in SD (417+/-58 vs 1021+/-112 pg ml(-1)) and WKY (309+/-59 vs 1433+/-187 pg ml(-1)), and in SHR (853+/-96 vs 1879+/-229 pg ml(-1)). These results demonstrate that natriuretic peptides mediate the urinary actions of moxonidine through natriuretic peptide receptors.

The cardiovascular and renal effects of a highly potent mu-opioid receptor agonist, cyclo[N epsilon,N beta-carbonyl-D-Lys2,Dap5]enkephalinamide.

Investigation of the acute cardiovascular and renal effects of cyclo[Nepsilon,Nbeta-carbonyl-D-Lys2,Dap5]enkephalinamide (cUENK6), the most potent mu-opioid receptor agonist, revealed dose-related effects, but most pronounced during the first hour post i.v. injections. During first hour, cUENK6 (3 microg/rat) stimulated (P<0.001) excretion of urine (1.1+/-0.2 vs. 3.3+/-0.3 ml/h), sodium (60+/-10 vs. 124+/-12 microeq/h), potassium and cGMP (1.76+/-0.19 vs. 4.92+/-0.80 nmol/h). These effects were inhibited by naloxone (4 mg/kg i.v.), but not by naloxonazine (35 mg/kg s.c.), or 4 mg/kg i.v. naloxone methiodide. cUENK6 stimulated urinary atrial natriuretic peptide (ANP)-like activity (113+/-12 vs. 167+/-20 pg/h, P<0.02) and the effect was totally abolished by naloxone. cUENK6 also suppressed the transient stress-induced elevation in blood pressures and heart rate that occurred over the first 30-min post-injection, an effect attenuated by naloxone. Plasma ANP increased 2-h post-injection (123+/-11 vs. 192+/-21 pg/ml, P<0.005), and was associated with augmented ANP mRNA levels in right atria and left ventricles. Thus, cUENK6 evokes renal effects by enhancing activity of the renal natriuretic peptide system.

Imidazoline receptors but not alpha 2-adrenoceptors are regulated in spontaneously hypertensive rat heart by chronic moxonidine treatment.

We have recently identified imidazoline I(1)-receptors in the heart. In the present study, we tested regulation of cardiac I(1)-receptors versus alpha(2) -adrenoceptors in response to hypertension and to chronic exposure to agonist. Spontaneously hypertensive rats (SHR, 12-14 weeks old) received moxonidine (10, 60, and 120 microg/kg/h s.c.) for 1 and 4 weeks. Autoradiographic binding of (125)I-paraiodoclonidine (0.5 nM, 1 h, 22 degrees C) and inhibition of binding with epinephrine (10(-10)-10(-5) M) demonstrated the presence of alpha(2)-adrenoceptors in heart atria and ventricles. Immunoblotting and reverse transcription-polymerase chain reaction identified alpha(2A)-alpha(2B)-, and alpha(2C), and -adrenoceptor proteins and mRNA, respectively. However, compared with normotensive controls, cardiac alpha(2) -adrenoceptor kinetic parameters, receptor proteins, and mRNAs were not altered in SHR with or without moxonidine treatment. In contrast, autoradiography showed that up-regulated atrial I(1)-receptors in SHR are dose-dependently normalized by 1 week, with no additional effect after 4 weeks of treatment. Moxonidine (120 microg/kg/h) decreased B(max) in right (40.0 +/- 2.9-7.0 +/- 0.6 fmol/unit area; p < 0.01) and left (27.7 +/- 2.8-7.1 +/- 0.4 fmol/unit area; p < 0.01) atria, and decreased the 85- and 29-kDa imidazoline receptor protein bands, in right atria, to 51.8 +/- 3.0% (p < 0.01) and 82.7 +/- 5.2% (p < 0.03) of vehicle-treated SHR, respectively. Moxonidine-associated percentage of decrease in B(max) only correlated with the 85-kDa protein (R(2) = 0.57; p < 0.006), suggesting that this protein may represent I(2)-receptors. The weak but significant correlation between the two imidazoline receptor proteins (R(2) = 0.28; p < 0.03) implies that they arise from the same gene. In conclusion, the heart possesses I(1)-receptors and alpha(2)-adrenoceptors, but only I(1)-receptors are responsive to hypertension and to chronic in vivo treatment with a selective I(1)-receptor agonist.

Chronic imidazoline receptor activation in spontaneously hypertensive rats.

BACKGROUND: Acute intravenous administration of moxonidine, an imidazoline I1-receptor agonist, reduces blood pressure (BP) in normotensive and hypertensive rats, induces diuresis and natriuresis, and stimulates plasma atrial natriuretic peptide (ANP). In these studies we investigated the involvement of natriuretic peptides (ANP and brain natriuretic peptide) in the effects of chronic activation of imidazoline receptors. METHODS: Spontaneously hypertensive rats (SHR; 12 to 14 weeks old) received 7-day moxonidine treatment at various doses (10, 20, 60, and 120 microg/kg/h) via subcutaneously implanted osmotic minipumps. RESULTS: Hemodynamic parameters (continuously monitored by telemetry) revealed that, compared with saline-treated rats, moxonidine dose-dependently decreased blood pressures (BPs). Maximal blood pressure lowering effect was achieved by day 4 of treatment, at which point 60 microg/kg/h reduced mean arterial pressure (MAP) by 14.5 +/- 6.8 mm Hg as compared with basal levels. The decrease in MAP was influenced by a drop in both diastolic and systolic pressures. Moxonidine treatment did not alter daily urinary sodium and potassium excretions, but 120 microg/kg/h moxonidine decreased urine volume after 2 days and increased cyclic guanosine 3'5'monophosphate excretion on days 4 to 7 of treatment. Chronic moxonidine treatment dose-dependently increased plasma ANP to reach, at 120 microg/kg/h, a 40% increase (P < .01) above that of corresponding saline-treated SHR, with a concomitant increase in left and right atrial ANP mRNA (more than twofold). Plasma BNP increased by 120 microg/kg/h moxonidine (11.0 +/- 1.1 v 16.5 +/- 1.9 pg/mL, P < .002) without significant increases in atrial and ventricular BNP mRNA. CONCLUSIONS: ANP and BNP may be involved in the antihypertensive effect of chronic moxonidine treatment. Accordingly, natriuretic peptides may contribute to the sympatholytic and cardioprotective effects of chronic activation of imidazoline I1-receptors.

Imidazoline receptors in the heart: characterization, distribution, and regulation.

Imidazoline receptors were identified in cardiac tissues of various species. Imidazoline receptors were immunolocalized in the rat heart. Membrane binding and autoradiography on frozen heart sections using 0.5 nM para-iodoclonidine (125I-PIC) revealed that binding was equally and concentration-dependently inhibited by epinephrine and imidazole-4-acetic acid (IAA), implying 125I-PIC binding to cardiac alpha2-adrenergic and I1-receptors, respectively. After irreversible blockade of alpha2-adrenergic receptors, binding was inhibited by the selective I1-agonist, moxonidine, and the I1-antagonist, efaroxan, in a concentration-dependent (10-12 to 10-5 M) manner. Calculation of kinetic parameters revealed that in canine left and right atria, I1-receptor Bmax was 13.4 +/- 1.7 and 20.1 +/- 3.0 fmol/mg protein, respectively. Compared to age-matched normotensive Wistar Kyoto rats, I1-receptors were increased in 12-week-old hypertensive rat (SHR) right (22.6 +/- 0.3 to 43.7 +/- 4.4 fmol/unit area, p < 0.01) and left atria (13.3 +/- 0.6 to 30.2 +/- 4.1 fmol/unit area, p < 0.01). Also, compared to corresponding normal controls, Bmax was increased in hearts of hamsters with advanced cardiomyopathy (13.9 +/- 0.4 to. 26.0 +/- 2.3 fmol/unit area, p < 0.01) and in human ventricles with heart failure (12.6 +/- 1.3 to 35.5 +/- 2.9 fmol/mg protein, p < 0.003). These studies demonstrate that the heart possesses imidazoline I1-receptors that are up-regulated in the presence of hypertension or heart failure, which would suggest their involvement in cardiovascular regulation.